Taxonomy and Quorum Quenching Potential of Reyranella spp. Isolated from Riverside Soil

국문 초록 (Abstract)

본 연구는 Reyranella 균주의 분류학적 특성 및 N-acyl-homoserine lactone (AHL)에 기반한 quorum sensing(QS) system의 길항작용인 quorum quenching(QQ)능력을 가진 Reyranella 균주에 대한 최초의 연구이다. 토양 시료의 옥천의 보청천의 3 장소에서 수행 되었으며, 총 분리한 182종 중 Reyranella는 3종이 발굴 되었다. Reyranella 속에 속하는 5개의 종과 분리한 3종을 대상으로 농도별 QQ 능력을 확인한 결과 모든 8개의 균에서 QQ활성을 확인 할 수 있었다. 이는 Reyranella의 QQ 능력이 유전자 수준에서 보존 되었을 가능성을 보여준다. 발굴된 3종 모두 QQ능력을 가지고 있었지만, 그 중 QQ 활성이 가장 좋으며 생장조건이 뛰어난 Reyranella MMS21-HV4-11T를 대상으로 연구를 수행하였다. Whole genome 분석을 통해 MMS21-HV4-11T의 AHL degradation enzyme에 연관된 3개의 유전자를 찾을 수 있었고, 이는 protein structure를 예상 수 있는 프로그램을 통해 N-acyl homoserine lactonase 2개와 N-acyl homoserine acylase 1개로 추정 및 구조적 확인을 하였다. 병원균의 QS을 억제할 수 있는지 보기 위해 Pectobactrium carotovorum supsp. carotovorum의 soft rot 억제능력 과 Pseudomonas aeruginosa QS작용 중 하나인 Swarming motility를 억제하는 것을 확인하여 간접적으로 biofilm형성 억제의 가능성을 확인하였다. MMS21-HV4-11T은 최근연종과 orthoANI 와 GGDC에서 각각 90, 39이하의 값을 보여 신종이며, 본 연구를 통해 MMS21-HV4-11T의 분류학적 특성을 확인하였다.

다국어 초록 (Multilingual Abstract)

A novel Gram-negative, rod shaped bacterial strain MMS21-HV4-11T displaying N-hexanoyl-L-homoserine lactone (C6-HSL)-degrading potential was isolated from riverside soil. The strain was found to be closely related to members of the genus Reyranella and showed 16S rRNA gene sequence similarities of 98.74, 97.87, and 97.80% with R. aquatilis seoho-37T, R. soli KIS14-15T and R. massiliensis 521T, respectively. Reyranella genus currently contains 5 species, and the quorum quenching activity of the type strains of 5 known species along with the isolate was tested. All strains of Reyranella were capable of degrading C6-HSL, thus showing quorum quenching potential at the genus level. Decomposed C6-HSL showed restoration from some concentrations under acidic conditions. The genome analysis indicated that strain MMS21-HV4-11T was found to have two lactonase and one acylase candidate genes, and SWISS MODEL confirmed the expected protein structures. In the quorum sensing inhibition assay using a plant pathogen Pectobacterium carotovorum subsp. carotovorum, the development of soft rot was significantly inhibited by MMS21-HV4-11T. In addition, the swarming motility by Pseudomonas aeruginosa was also significantly inhibited. Since the isolate did not display direct antibacterial activity against both species, this inhibition was certainly due to the quorum quenching activity by MMS21-HV4-11T. This is the first study on quorum quenching activity from Reyranella, and MMS21-HV4-11T was confirmed as a new species through chemotaxonomic and phylogenomic characterization, for which a new species Reyranella okcheonensis sp. nov. is proposed (type strain=MMS21-HV4-11T =KCTC 82780T =LMG 32365T).

목차 (Table of Contents)

• 1. Abstract 1
• 2. Introduction 2
• 3. Materials and Methods 5
• 3.1. Sampling sites and isolation 5
• 3.2. Identification and selection based on 16S rRNA gene 7
• 3.2.1. Identification of strains based on 16S rRNA gene. 7
• 3.2.2. Phylogenetic analysis based on 16S rRNA gene. 8
• 3.3. Quorum Quenching (QQ) activity 9
• 3.3.1. Measurement of QQ activity 9
• 3.3.2. QQ assay for Reyranella spp. 10
• 3.3.3. C6-HSL restoration by acidification. 11
• 3.3.4. Detection of AHL degradation activity by strain MMS21-HV4-11T 11
• 3.4. Quorum Sensing (QS) inhibition assay. 13
• 3.4.1. Antagonism assay 13
• 3.4.2. Soft rot inhibition assay 13
• 3.4.3. Swarming motility assay 14
• 3.5. Whole genome analysis 15
• 3.5.1. Whole genome sequence analyses. 15
• 3.5.2. Rapid annotation using subsystem Technology (RAST) & Prokaryotic Genome Annotation Pipeline (PGAP) 15
• 3.6. Phylogenetic analysis based on N-acyl homoserine lactone degrading protein sequence 16
• 3.7. Potential enzyme structure analysis 17
• 3.7.1. HXHXDH BOX structural analysis of N-acyl homoserine lactonase 17
• 3.7.2. Prediction of overall structures 18
• 3.7.3. Prediction of active sites of N-acyl homoserine lactone degrading enzymes 19
• 3.8. Taxonomic characterization 20
• 3.8.1. Morphology 20
• 3.8.2. Optimal growth condition 20
• 3.8.3. API test 21
• 3.8.4. Catalase and oxidase test 21
• 3.8.5. Scanning electron microscope 21
• 3.8.6. Cellular fatty acid analysis 22
• 3.8.7. Polar lipid analysis 22
• 3.8.8. Respiratory isoprenoid quinone analysis 23
• 3.8.9. Comparative genome analysis 23
• 4. Results 24
• 4.1. Total strains isolated from Bocheong stream in Okcheon 24
• 4.2. QQ ability of isolates from Bocheong stream 26
• 4.2.1. QQ ability of total isolates 26
• 4.2.2. Phylogenetic analysis of Reyranella spp. based on 16S rRNA gene 32
• 4.2.3. QQ ability of Reyranella spp 34
• 4.2.4. Degradation of AHLs by strain MMS21-HV4-11T 36
• 4.3. QS inhibition assay using plant pathogens 38
• 4.3.1. Antagonism 38
• 4.3.2. Effect of the QQ strain MMS21-HV4-11T against Pcc 40
• 4.3.3. Inhibition of swarming motility by P. aeruginosa PA14 43
• 4.4. Prediction of C6-HSL degradation enzyme 45
• 4.4.1. Whole genome information 45
• 4.4.2. Rapid Annotation using Subsystem Technology 45
• 4.5. Phylogenetic analysis based on protein sequence 48
• 4.5.1. N-acyl homoserine lactonase group 48
• 4.5.2. N-acyl homoserine lactone acylase group 48
• 4.6. Structural analysis of MMS21-HV4-11T potential enzymes 51
• 4.6.1. HXHXDH BOX in N-acyl homoserine lactonase 51
• 4.6.2. Overall structure analysis using SWISS-MODEL. 54
• 4.6.3. Active site prediction 54
• 4.7. Description of Reyranella okcheonensis sp. nov. 58
• 5. Discussion 66
• 6. References 70
• SUMMARY (in Korean) 74